mRNA Deep Sequencing Research Articles

Abstract We081: β1 integrins regulate cellular behavior and cardiomyocyte organization during ventricular wall formation

Introduction: Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. However, the mechanisms through which these cells organize themselves to establish the tissue architecture of trabeculae and the ventricular wall remain inadequately elucidated. Integrins are the primary cell surface receptors for adhesion to the ECM and mediate both inside-out and outside-in signal transduction pathways that control various cell functions, including proliferation, survival, migration, and gene expression. We found that the β1 integrin subunit (β1), encoded by Itgb1 , is highly expressed in all cardiac cell types. Hypothesis: β1 integrins play a crucial role in regulating cardiomyocyte behavior and organization during ventricular wall morphogenesis in mice. Methods and Results: We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/β integrins and their ligands in the embryonic heart. β1 and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1 , which encodes the β1, was deleted via Nkx2.5 Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts lack a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of β1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. Conclusions: β1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. β1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of β1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.

β1 integrins regulate cellular behaviour and cardiomyocyte organization during ventricular wall formation.

The mechanisms regulating the cellular behaviour and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. This study aims to determine whether and how β1 integrins regulate cardiomyocyte behaviour and organization during ventricular wall morphogenesis in the mouse. We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/β integrins and their ligands in the embryonic heart. Integrin β1 subunit (β1) and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1, which encodes the β1, was deleted via Nkx2.5Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts display an absence of a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of β1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. β1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. β1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of β1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.

Comparative Analyses of Reproductive Caste Types Reveal Vitellogenin Genes Involved in Queen Fertility in Solenopsis invicta.

The red imported fire ant (Solenopsis invicta Buren) is a social pest species with a robust reproductive ability that causes extensive damage. Identification of the genes involved in queen fertility is critical in order to better understand the reproductive biology and screening for the potential molecular targets in S. invicta. Here, we used the mRNA deep sequencing (RNA-seq) approach to identify differentially expressed genes (DEGs) in the transcriptomes of three reproductive caste types of S. invicta, including queen (QA) and winged female (FA) and male (MA) ants. The genes that were specific to and highly expressed in the queens were then screened, and the Vg2 and Vg3 genes were chosen as targets to explore their functions in oogenesis and fertility. A minimum of 6.08 giga bases (Gb) of clean reads was obtained from all samples, with a mapping rate > 89.78%. There were 7524, 7133, and 977 DEGs identified in the MA vs. QA, MA vs. FA, and FA vs. QA comparisons, respectively. qRT-PCR was used to validate 10 randomly selected DEGs, including vitellogenin 2 (Vg2) and 3 (Vg3), and their expression patterns were mostly consistent with the RNA-seq data. The S. invicta Vgs included conserved domains and motifs that are commonly found in most insect Vgs. SiVg2 and SiVg3 were highly expressed in queens and winged females and were most highly expressed in the thorax, followed by the fat body, head, and epidermis. Evaluation based on a loss-of-function-based knockdown analysis showed that the downregulation of either or both of these genes resulted in smaller ovaries, less oogenesis, and less egg production. The results of transcriptional sequencing provide a foundation for clarifying the regulators of queen fertility in S. invicta. The functions of SiVg2 and SiVg3 as regulators of oogenesis highlight their importance in queen fecundity and their potential as targets of reproductive disruption in S. invicta control.

The miR-221/222 cluster regulates hematopoietic stem cell quiescence and multipotency by suppressing both Fos/AP-1/IEG pathway activation and stress-like differentiation to granulocytes.

Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations.

MicroRNA and mRNA sequencing analyses reveal key hepatic metabolic and signaling pathways responsive to maternal undernutrition in full-term fetal pigs

Maternal undernutrition is highly prevalent in developing countries, leading to severe fetus/infant mortality, intrauterine growth restriction, stunting, and severe wasting. However, the potential impairments of maternal undernutrition to metabolic pathways in offspring are not defined completely. In this study, 2 groups of pregnant domestic pigs received nutritionally balanced gestation diets with or without 50% feed intake restriction from 0 to 35 gestation days and 70% from 35 to 114 gestation days. Full-term fetuses were collected via C-section on day 113/114 of gestation. MicroRNA and mRNA deep sequencing were analyzed using the Illumina GAIIx system on fetal liver samples. The mRNA-miRNA correlation and associated signaling pathways were analyzed via CLC Genomics Workbench and Ingenuity Pathway Analysis Software. A total of 1189 and 34 differentially expressed mRNA and miRNAs were identified between full-nutrition (F) and restricted-nutrition (R) groups. The correlation analyses showed that metabolic and signaling pathways such as oxidative phosphorylation, death receptor signaling, neuroinflammation signaling pathway, and estrogen receptor signaling pathways were significantly modified, and the gene modifications in these pathways were associated with the miRNA changes induced by the maternal undernutrition. For example, the upregulated (P<.05) oxidative phosphorylation pathway in R group was validated using RT-qPCR, and the correlational analysis indicated that miR-221, 103, 107, 184, and 4497 correlate with their target genes NDUFA1, NDUFA11, NDUFB10 and NDUFS7 in this pathway. These results provide the framework for further understanding maternal malnutrition's negative impacts on hepatic metabolic pathways via miRNA-mRNA interactions in full-term fetal pigs.

Ribosomal Profiling by Gradient Fractionation of Cell Lysates.

Ribosomal profiling is a widely used technique for deep sequencing of ribosome-protected mRNA and for measuring ribosome status in cells. It is a powerful method that is typically employed for monitoring and measuring protein translation status and ribosome activity. Also, it has been used for monitoring the ribosomal stress-responsive events in the ribosome activity. Furthermore, this approach enables understanding of translational regulation, which is invisible in most proteomic approaches. Moreover, this method is known as an important approach for biological discovery such as identification of translation products. Hence, this methodology will be useful for studying cellular events engaging in ribosome assembly, ribosome biogenesis, ribosome activity, translation during the cell cycle, cell proliferation, and growth as well as the ribosomal stress response in mammalian cells.

The dynamics of molecular, immune and physiological features of the host and the gut microbiome, and their interactions before and after onset of laying in two hen strains

Aggregation of data, including deep sequencing of mRNA and miRNA data in jejunum mucosa, abundance of immune cells, metabolites, or hormones in blood, composition of microbiota in digesta and duodenal mucosa, and production traits collected along the lifespan, provides a comprehensive picture of lifelong adaptation processes. Here, respective data from two laying hen strains (Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL) collected at 10, 16, 24, 30, and 60 wk of age were analyzed. Data integration revealed strain- and stage-specific biosignatures, including elements indicative of molecular pathways discriminating the strains. Although the strains performed the same, they differed in the activity of immunological and metabolic functions and pathways and showed specific gut-microbiota-interactions in different production periods. The study shows that both strains employ different strategies to acquire and maintain their capabilities under high performance conditions, especially during the transition phase. Furthermore, the study demonstrates the capacity of such integrative analyses to elucidate molecular pathways that reflect functional biodiversity. The bioinformatic reduction of the multidimensional data provides good guidance for further manual review of the data.

Early Transcriptional Changes of Adipose-Derived Stem Cells (ADSCs) in Cell Culture.

Background and Objectives: While autologous fat grafting has been carried out in the clinical field for many years, the utilization of isolated and cultured adipose-derived stem cells (ADSCs) is highly restricted in many countries. However, ADSCs are under investigation currently and heavily researched in many cell-based therapy approaches in the field of regenerative medicine. Objective: For the utilization of future cell-based therapies with ADSCs, in vitro cell expansion might be necessary in many cases. Thus, the cellular characteristics of ADSCs may be altered though the process of being cultured. The aim of this study was to assess changes in the gene expression profile of ADSCs after cell expansion for 48 h. Materials and Methods: Isolated ADSCs from five different donors were used for in vitro expansion. For the evaluation of the gene expression profile, mRNA deep Next-Generation Sequencing was performed to evaluate the differences between cultured and freshly isolated cells. Results: Our study gives insight into transcriptional changes in ADSCs after a short cell cultivation period. This includes the most prominent upregulated genes such as PPL, PRR15, CCL11 and ABCA9, as well the most downregulated genes, which are FOSB, FOS, EGR1 and DUSP6. Furthermore, we showed different biological processes that changed during short-term cell expansion, which led to downregulation of fat-associated metabolism hormone processes and to an upregulation of extracellular matrix-associated genes. Conclusion: In conclusion, our study reveals a detailed insight into early changes in the gene expression profile of cultured ADSCs. Our results can be utilized in future experiments.

Role of Proximal Tubule DPP4 in Inflammation and Kidney Disease Progression

Obesity and overweight leads to an entity called obesity related kidney disease (ORKD), which is a major risk factor for chronic kidney disease (CKD) progression to End Stage Renal Disease and/or dialysis. ORKD is characterized by proximal tubule brush border vacuolization and injury, basement membrane thickening, glomerular accumulation of fat globules and glomerulomegaly. We have developed a mouse model of Western diet (high fat and high sucrose)‐induced obesity that manifests all the features of ORKD. In addition, our lab observed that obese mice fed a Western diet, manifest early hyperfiltration (3 months into feeding). It is later followed by reduction in glomerular filtration rate (GFR, beginning at 18 months of age), and this pattern mimics human diabetic kidney disease progression. Our lab also observed that in ORKD, there is increased Dipeptidyl Peptidase 4 (DPP4) activity in the kidney, especially the proximal tubules and proximal tubule specific deletion of DPP4 improves the GFR. Lastly, our lab demonstrated that there is a major role for inflammation in the obese kidney and DPP4 inhibitors mitigate the inflammation. However, it is not known if and how proximal tubule DPP4 contributes to inflammation and pathophysiological changes in ORKD. Therefore, we hypothesized that proximal tubule DPP4 mediates inflammation and contributes to the pathophysiological changes in ORKD, thereby identifying DPP4 as a target to prevent CKD progression.MethodsProximal tubule specific DPP4fl/fl;SGLT2Cre+ (PT DPP4KO) and DPP4fl/fl;SGLT2Cre‐ (PT DPP4WT) mice were fed a WD and control chow (CD) for 6 mths, 15 mths and till sacrifice (18 to 27 mths). Inflammation was assessed both by flow cytometry assessment of kidney immune cells and expression of pro and anti‐inflammatory genes and proteins).ResultsThere were more pro‐inflammatory as well as DPP4 expressing macrophages in the WD‐fed WT group compared to the KO group (p&lt;0.05). There was also activation of the T‐cells in particular the CD8s which was suppressed in the KO group (p&lt;0.05). Deep sequencing of kidney mRNA revealed activation of pro‐inflammatory cytokines including Th1, Th17, M1 macrophage‐based cytokines and TLR4 and ICAM‐1. The KO group showed improvement in both TLR4 and ICAM‐1 (p&lt;0.05). At the protein level, there was an increase in pro‐fibrotic (Desmin) and pro‐inflammatory (ERK) proteins in the WD‐fed WT groups and phosphorylation of ERK was suppressed in the KO group (p&lt;0.05). When inflammation was assessed across the months it seemed that the earliest changes were in the growth pathways and beta‐oxidation of lipids, followed by the more inflammatory genes later in the course of the disease.ConclusionWD feeding led to hyperfiltration followed by a decline in GFR that was partly mitigated by PT deletion of DPP4 and this was associated with improved kidney histology and less fibrosis. WD feeding in WT mice was associated with increase in expression of several pro inflammatory genes and TLR4 and ICAM 1 were normalized in KO mice. DPP4 protein expression in kidney was associated with increased phosphorylation of ERK and this was suppressed in KO mice. Overall, these data suggest that suppression of early activation of DPP4 in ORKD may slow down the progression of CKD.

Design, synthesis, and evaluation of fluoroquinolone derivatives as microRNA-21 small-molecule inhibitors

MicroRNA-21 (miRNA-21) is highly expressed in various tumors. Small-molecule inhibition of miRNA-21 is considered to be an attractive novel cancer therapeutic strategy. In this study, fluoroquinolone derivatives A1–A43 were synthesized and used as miRNA-21 inhibitors. Compound A36 showed the most potent inhibitory activity and specificity for miRNA-21 in a dual-luciferase reporter assay in HeLa cells. Compound A36 significantly reduced the expression of mature miRNA-21 and increased the protein expression of miRNA-21 target genes, including programmed cell death protein 4 (PDCD4) and phosphatase and tensin homology deleted on chromosome ten (PTEN), at 10 μM in HeLa cells. The Cell Counting Kit-8 assay (CCK-8) was used to evaluate the antiproliferative activity of A36; the results showed that the IC50 value range of A36 against six tumor cell lines was between 1.76 and 13.0 μM. Meanwhile, A36 did not display cytotoxicity in BEAS-2B cells (lung epithelial cells from a healthy human donor). Furthermore, A36 significantly induced apoptosis, arrested cells at the G0/G1 phase, and inhibited cell-colony formation in HeLa cells. In addition, mRNA deep sequencing showed that treatment with A36 could generate 171 dysregulated mRNAs in HeLa cells, while the expression of miRNA-21 target gene dual-specificity phosphatase 5 (DUSP5) was significantly upregulated at both the mRNA and protein levels. Collectively, these findings demonstrated that A36 is a novel miRNA-21 inhibitor.

Human type 2 diabetes mellitus-associated transcriptional disturbances in a high-sugar diet long-term exposed Drosophila melanogaster

Type 2 Diabetes mellitus (T2DM) is a multifactorial and polygenic disorder with the molecular bases still idiopathic. Experimental analyses and tests are quite limited upon human samples due to the access, variability of patient's conditions, and the size and complexity of the genome. Therefore, high-sugar diet exposure is commonly used for modeling T2DM in non-human animals, which includes invertebrate organisms like the fruit fly Drosophila melanogaster. Interestingly, high-sugar diet (HSD) induces delayed time for pupation and reduced viability in fruit fly larvae hatched from a 30% sucrose-containing medium (HSD-30%). Here we carried out an mRNA-deep sequencing study to identify differentially transcribed genes in adult fruit fly hatched and reared from an HSD-30%. Seven days after hatching, flies reared on control and HSD-30% were used to glucose and triglyceride level measurements and RNA extraction for sequencing. Remarkably, glucose levels were about 2-fold higher than the control group in fruit flies exposed to HSD-30%, whereas triglycerides levels increased 1.7-fold. After RNA-sequencing, we found that 13.5% of the genes were differentially transcribed in the dyslipidemic and hyperglycaemic insects. HSD-30% up-regulated genes involved in ribosomal biogenesis (e.g. dTOR, ERK and dS6K) and down-regulated genes involved in energetic process (e.g. Pfk, Gapdh1, and Pyk from pyruvate metabolism; kdn, Idh and Mdh2 from the citric acid cycle; ATPsynC and ATPsynẞ from ATP synthesis) and insect development. We found a remarkable down-regulation for Actin (Act88F) that likely impairs muscle development. Moreover, HSD-30% up-regulated both the insulin-like peptides 7 and 8 and down-regulated the insulin receptor substrate p53, isoform A and insulin-like peptide 6 genes, whose functional products are insulin signaling markers. All these features pointed together to a tightly correlation of the T2DM-like phenotype modeled by the D. melanogaster and an intricate array of phenomena, which includes energetic processes, muscle development, and ribosomal synthesis as that observed for the human pathology.

MicroRNA and mRNA Sequencing Analyses Reveal Key Hepatic Metabolic Pathways Responsive to Maternal Malnutrition in Full-Term Fetal Pigs

ObjectivesMaternal and infant undernutrition is highly prevalent in developing countries, leading to serious fetus/infant mortality, intrauterine growth restriction, stunting, and severe wasting. However, the effects of maternal undernutrition have generally focused on the reduced maternal nutrient supply to the fetus. The potential impairment of fetal metabolic pathways has not been well studied. MethodsPregnant gilts (Landrace x Yorkshire x Duroc) received the NRC gestation diet with (n = 4) or without (n = 4) 50% intake restriction at insemination day and 70% for the following gestation period. Full term fetuses were obtained via C-section, two piglets were selected from each gilt in both groups and subject to hepatic tissue collection. MicroRNA and mRNA deep sequencing analysis was performed using the Illumina GAIIx system. The mRNA-miRNA correlation and associated signaling pathways were analyzed via CLC workbench, Ingenuity Pathway Analysis Software. ResultsA total of 42 differentially expressed miRNAs were identified between intake-restriction and full-nutrition group. Among of these, mir-206, mir-133b, mir-1246, mir-1843 and mir-7139 are the most downregulated and mir-10b, mir-708 and mir-222 are the most upregulated miRNAs. A total of 1215 mRNAs were identified to differentially expressed between two groups. Two metabolic pathways: retinol biosynthesis and oxidative phosphorylation were significantly modified, and the modification was associated with the miRNA changes induced by the maternal feed restriction. Briefly, the retinol biosynthesis pathway was upregulated (p < 0.01), in which those differential expressed mir-221, mir-4492, mir-1281 and mir-4492 were predicted targeting genes AADAC, CES3, PNPLA3 and RDH13 in the pathway. The oxidative phosphorylation pathway was upregulated (p < 0.05), and those differential expressed mir-1843, mir-222 and mir-184 were predicted targeting genes ATP5F1C, NDUFA1, NDUFB10, and NDUFS7 in this pathway. ConclusionsThese results provide the framework for further understanding of negative impact of maternal malnutrition on hepatic metabolic pathways via miRNA-RNA interactions in full-term fetal pigs. Funding SourcesSupported in part by the Bill and Melinda Gates Foundation (GCE OPP1061037) and by the North Carolina Agricultural Research Service.

MicroRNA-497 Reduction and Increase of Its Family Member MicroRNA-424 Lead to Dysregulation of Multiple Inflammation Related Genes in Synovial Fibroblasts With Rheumatoid Arthritis.

ObjectivesMounting evidence has demonstrated that microRNAs (miRNAs) participate in rheumatoid arthritis (RA). The role of highly conserved miR-15/107 family in RA has not been clarified yet, and hence investigated in this study.MethodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to evaluate the expression of miRNAs and genes. Cell counting kit 8 (CCK-8) and FACS were used to detect proliferation and apoptosis. Protein expression was detected by using Western blotting. mRNA deep sequencing and cytokine antibody array were used to analyze differentially expressed genes, signaling pathways and cytokines.ResultsThe expression of miR-15a, miR-103, miR-497, and miR-646 was found decreased, while miR-424 increased in RA patients. MiR-424 and miR-497 were further investigated and the results showed that they could regulate the expression of multiple genes in rheumatoid arthritis synovial fibroblast (RASF) and affect signaling pathways. At the protein level, miR-497 mimic altered all the selected inflammation-related genes while miR-424 inhibitor only affected part of genes. MiR-497 mimic, rather than miR-424 inhibitor, had significant effects on proliferation and apoptosis of RASF. DICER1 was found to positively regulate the expression of miR-424 and miR-497, while DICER1 was also negatively regulated by miR-424. The increase of miR-424 could reduce miR-497 expression, thus forming a loop, which facilitated explaining the dysregulated miR-424 and miR-497 in RA.ConclusionThe miR-424 and miR-497 of miR-15/107 family affect cell proliferation and apoptosis in RA, and the proposed miR-424-DICER1-miR-497 feedback loop provides a novel insight into regulating miRNA expression and a candidate target for controlling RA.

Integrated analysis of miRNAome and transcriptome reveals miRNA-mRNA network regulation in Vibrio alginolyticus infected thick shell mussel Mytilus coruscus

The thick shell mussel Mytilus coruscus has developed into a model species for studying the interaction between molluscs and environmental stimuli. Herein, integrated analysis of miRNAome and transcriptome was performed to reveal miRNA-mRNA network regulation in Vibrio alginolyticus infected M. coruscus. There have detected some histological abnormalities in digestive gland and gills of V. alginolyticus challenged mussels, ascertaining the effective irritation by the present bacterial strain. A total of 265 novel miRNAs were finally predicted, of which 26 were differentially expressed miRNAs (DEMs). Additionally, 667 differentially expressed genes (DEGs) were detected, which may be potentially associated with innate immune response to V. alginolyticus infection. A regulatory network linked to 22 important pathways and 16 DEMs and 34 OGs was constructed. Some traditional immune-related signaling pathways such as toll-like receptor signaling pathway (TLR) signaling pathway, transforming growth factor-beta (TGF-beta) signaling pathway, peroxisome, phagosome, lysosome, mammalian target of rapamyoin (mTOR) signaling pathway were linked to specific miRNAs and genes in this network. Further, interactional relationship between certain miRNAs and TLR pathway was dissected, which the results predicted that a number of TLRs and TLR-associated signaling genes including TLR1, TLR2, TLR4, TLR6, IRAK1, TRAF6, MAPK, and IL-17 were negatively regulated by novel_miR_11, novel_miR_145, novel_miR_196, novel_miR_5, novel_miR_163 and novel_miR_217 in the TLR pathway. Additionally, interactional relationship between novel_miR_145 and TLR2 was validated by laboratory experiment. The integrated analysis of mRNA and microRNA deep sequencing data exhibited a sophisticated miRNA-mRNA regulation network in M. coruscus in response to V. alginolyticus challenge, which shed a new light on the underlying mechanism of molluscan confronting bacterial infection.

Genomic analysis of a second rainbow trout line (Arlee) leads to an extended description of the IGH VDJ gene repertoire

High-throughput sequencing technologies brought a renewed interest for immune repertoires. Fish Ab and B cell repertoires are no exception, and their comprehensive analysis can both provide new insights into poorly understood immune mechanisms, and identify markers of protection after vaccination. However, the lack of genomic description and standardized nomenclature of IG genes hampers accurate annotation of Ig mRNA deep sequencing data. Complete genome sequences of Atlantic salmon and rainbow trout (Swanson line) recently allowed us to establish a comprehensive and coherent annotation of Salmonid IGH genes following IMGT standards. Here we analyzed the IGHV, D, and J genes from the newly released genome of a second rainbow trout line (Arlee). We confirmed the validity of salmonid IGHV subgroups, and extended the description of the rainbow trout IGH gene repertoire with novel sequences, while keeping nomenclature continuity. This work provides an important resource for annotation of high-throughput Ab repertoire sequencing data.

The Target MicroRNAs and Potential Underlying Mechanisms of Yiqi-Bushen-Tiaozhi Recipe against-Non-Alcoholic Steatohepatitis.

MicroRNAs (miRNAs) have emerged as potential therapeutic targets for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). Traditional Chineses Medicine (TCM) plays an important role in the prevention or treatment of NAFLD/NASH. However, miRNA targets of TCM against NASH still remain largely unknown. Here, we showed that Yiqi-Bushen-Tiaozhi (YBT) recipe effectively attenuated diet-induced NASH in C57BL/6 mice. To identify the miRNA targets of YBT and understand the potential underlying mechanisms, we performed network pharmacology using miRNA and mRNA deep sequencing data combined with Ingenuity Pathway Analysis (IPA). Mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7g-3p and mmu-miR-106b-3p were screened as the main targets of YBT. Our results suggested that YBT might alleviate NASH by regulating the expression of these miRNAs that potentially modulate inflammation/immunity and oxidative stress. This study provides useful information for guiding future studies on the mechanism of YBT against NASH by regulating miRNAs.

Using mRNA deep sequencing to analyze differentially expressed genes during Panax notoginseng saponin treatment of ischemic stroke.

Treatment with Panax notoginseng saponin (PNS) can prevent neurological damage in middle cerebral artery occlusion model rats to promote recovery after a stroke. However, the exact molecular mechanisms are unknown and require further study. In the present study, mRNA sequencing was employed to investigate differential gene expression between model and sham groups, and between model and PNS-treated groups. Enrichment of gene data was performed using Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes database. Hub genes were identified and networks were constructed using Cytoscape that were further verified by reverse transcription-quantitative PCR. A total of 1,104 genes of interest were found, which included 690 upregulated and 414 downregulated genes that were identified when the model was compared with the sham group. Additionally, 817 genes of interest, which included 390 upregulated and 427 downregulated genes, were identified when the PNS-treated group was compared with the model group. There were 303 overlapping genes of interest between the analysis of model to sham groups, and the analysis of model to PNS-treated groups. The top 10 genes from the 303 aberrantly expressed genes of interest included ubiquitin conjugating enzyme E2 variant 2, small ubiquitin-related modifier 1, small RNA binding exonuclease protection factor La, Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed, centrosomal protein 290 kDa, DNA-directed RNA polymerase II subunit K, cullin-4B, matrin-3 and vascular endothelial growth factor receptor 2. In conclusion, these genes may be important in the underlying mechanism of PNS treatment in ischemic stroke. Additionally, the present data provided novel insight into the pathogenesis of ischemic stroke.

Transcriptome and translatome profiles of Streptomyces species in different growth phases

Streptomyces are efficient producers of various bioactive compounds, which are mostly synthesized by their secondary metabolite biosynthetic gene clusters (smBGCs). The smBGCs are tightly controlled by complex regulatory systems at transcriptional and translational levels to effectively utilize precursors that are supplied by primary metabolism. Thus, dynamic changes in gene expression in response to cellular status at both the transcriptional and translational levels should be elucidated to directly reflect protein levels, rapid downstream responses, and cellular energy costs. In this study, RNA-Seq and ribosome profiling were performed for five industrially important Streptomyces species at different growth phases, for the deep sequencing of total mRNA, and only those mRNA fragments that are protected by translating ribosomes, respectively. Herein, 12.0 to 763.8 million raw reads were sufficiently obtained with high quality of more than 80% for the Phred score Q30 and high reproducibility. These data provide a comprehensive understanding of the transcriptional and translational landscape across the Streptomyces species and contribute to facilitating the rational engineering of secondary metabolite production.

Integrated miRNAome and Transcriptome Analysis Reveals Argonaute 2-Mediated Defense Responses Against the Devastating Phytopathogen Sclerotinia sclerotiorum.

Argonaute 2 (AGO2)-mediated role in plant defense against fungal pathogens remains largely unknown. In this study, integrated miRNAome and transcriptome analysis employing ago2 mutant was performed to reveal AGO2-associated miRNAs and defense responses against the devastating necrotrophic phytopathogen Sclerotinia sclerotiorum. Both miRNAome and transcriptomes of S. sclerotiorum-inoculated ago2-1 mutant (ago2-Ss) and wild-type (WT-Ss) as well as mock-inoculated ago2-1 mutant (ago2) and wild-type (WT) Arabidopsis plants, were analyzed by sRNA and mRNA deep sequencing. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) of the comparisons WT-Ss/WT, ago2/WT, ago2-Ss/WT-Ss, and ago2-Ss/ago2 were identified. Furthermore, integration analysis for the DEMs and DEGs identified over 40 potential AGO2-dependent Sclerotinia sclerotiorum-responsive (ATSR) DEM-DEG pairs involving modulation of immune recognition, calcium flux, redox homeostasis, hormone accumulation and signaling, cell wall modification and metal ion homeostasis. Data-mining result indicated that most of the DEMs were bound with AGO2. Moreover, Arabidopsis mutant analysis demonstrated that three ROS and redox homeostatasis related DEGs of identified DEM-DEG pairs, GSTU2, GSTU5, and RBOHF contributed to the AGO2-mediated defense against S. sclerotiorum. This work provides genome-wide prediction of miRNA–target gene pairs that are potentially associated with the AGO2-dependent resistance against S. sclerotiorum.

Multi-kinase control of environmental stress responsive transcription.

Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR)-a gene expression signature shared across the set of perturbations-in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is an upstream negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.